Tribonectins

ABSTRACT

The invention features a tribonectin and a method of tribosupplementation carried out by administering tribonectins directly to an injured or arthritic joint.

BACKGROUND OF THE INVENTION

The invention relates to lubrication of mammalian joints.

Osteoarthritis (OA) is the one of the most common form of joint disease.Factors which contribute to the development of OA include a familyhistory of OA, previous damage to the joint through injury or surgery,and age of the joint, i.e., “wear and tear” of the articulating surfacesof the joint. OA is very common in older age groups, but can affectchildren as well.

Current treatment is directed to relieving pain and other symptoms ofOA, e.g., by administering analgesics and anti-inflammatory drugs. Othertherapeutic approaches include viscosupplementation by administeringhyaluronic acid and derivatives thereof to joint tissue to increase theviscosity of synovial fluid.

SUMMARY OF THE INVENTION

The invention features a novel treatment for osteoarthritis and otherdegenerative joint diseases by tribosupplementation.Tribosupplementation is carried out by administering lubricatingpolypeptides directly to the injured or arthritic joint. Unlikeviscosupplementation, tribosupplementation does not substantiallyincrease the viscosity of the solution, e.g, synovial fluid, to which itis added. The viscosity of a solution to which a tribonectin is addedincreases no more than 10%, preferably no more than 5%, more preferablyno more than 2%, more preferably no more than 1%. Most preferably, theviscosity of the solution to which a tribonectin is added is unalteredor decreases.

Accordingly, the invention provides a tribonectin. A tribonectin is anartificial boundary lubricant which contains at least one repeat of anamino acid sequence which is at least 50% identical to KEPAPTT (SEQ IDNO:3). A tribonectin is formulated for administration to a mammalianjoint. Preferably, the tribonectin is a recombinant orchemically-synthesized lubricating polypeptide. For example, atribonectin includes a substantially pure polypeptide the amino acidsequence of which includes at least one but less than 76 subunits. Eachsubunit contains at least 7 amino acids (and typically, 10 or feweramino acids). The amino acid sequence of each subunit is at least 50%identical to SEQ ID NO:3, and a non-identical amino acid in thereference sequence is a conservative amino acid substitution. Forexample, one or both of the threonine residues are substituted with aserine residue. Preferably, the amino acid sequence of the subunit isidentical to SEQ ID NO:3. The tribonectin may also contain one or morerepeats of the amino acid sequence XXTTX (SEQ ID NO:4). Polypeptides orother compounds described herein are said to be “substantially pure”when they are within preparations that are at least 60% by weight (dryweight) the compound of interest. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight the compound of interest. Purity can be measured by anyappropriate standard method, for example, by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

Where a particular polypeptide or nucleic acid molecule is said to havea specific percent identity to a reference polypeptide or nucleic acidmolecule of a defined length, the percent identity is relative to thereference polypeptide or nucleic acid molecule. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It can also be a100 amino acid long polypeptide which is 50% identical to the referencepolypeptide over its entire length.

A polypeptide or nucleic acid molecule which is “substantiallyidentical” to a given reference polypeptide or nucleic acid molecule isa polypeptide or nucleic acid molecule having a sequence that has atleast 85%, preferably 90%, and more preferably 95%, 98%, 99% or moreidentity to the sequence of the given reference polypeptide sequence ornucleic acid molecule. “Identity” has an art-recognized meaning and iscalculated using well known published techniques, e.g., ComputationalMolecular Biology, 1988, Lesk A. M., ed., Oxford University Press, NewYork; Biocomputing: Informatics and Genome Projects, 1993, Smith, D. W.,ed., Academic Press, New York; Computer Analysis of Sequence Data, PartI, 1994, Griffin, A. M. and Griffin, H. G., eds., Humana Press, NewJersey; Sequence Analysis in Molecular Biology, 1987, Heinje, G.,Academic Press, New York; and Sequence Analysis Primer, 1991, Gribskov,M. and Devereux, J., eds., Stockton Press, New York). While there exista number of methods to measure identity between two polynucleotide orpolypeptide sequences, the term “identity” is well known to skilledartisans and has a definite meaning with respect to a given specifiedmethod. Sequence identity described herein is measured using theSequence Analysis Software Package of the Genetics Computer Group (GCS),University of Wisconsin Biotechnology Center, 1710 University Avenue,Madison, Wis. 53705), with the default parameters as specified therein.

A tribonectin is characterized as reducing the coefficient of friction(μ) between bearing surfaces. For example, reduction of friction ismeasured in vitro by detecting a reduction in friction in a frictionapparatus using latex:glass bearings. Reduction of friction is alsomeasured in vivo, e.g., by measuring reduction of patient pain.Tribonectins of the invention are lubricating compositions. Polypeptidesthat have at least 50% (but less than 100%) amino acid sequence identityto a reference sequence are tested for lubricating function by measuringa reduction in the μ between bearing surfaces.

A tribonectin includes an O-linked oligosaccharide, e.g., anN-acetylgalactosamine and galactose in the form β(1-3)Gal-GalNAC. Forexample, KEPAPTT (SEQ ID NO:3) and XXTTTX (SEQ ID NO:4) repeat domainsare glycosylated by β(1-3)Gal-GalNAC (which may at times be capped withNeuAc in the form of β(1-3)Gal-GalNAC-NeuAc. The term “glycosylated”with respect to a polypeptide means that a carbohydrate moiety ispresent at one or more sites of the polypeptide molecule. For example,at least 10%, preferably at least 20%, more preferably at least 30%, andmost preferably at least 40% of the tribonectin is glycosylated. Up to50% or more of the tribonectin can be glycosylated. Percentglycosylation is determined by weight.

A tribonectin polypeptide can contain a substantially pure fragment ofmegakaryocyte stimulating factor (MSF). For example, the molecularweight of a substantially pure tribonectin having an amino acid sequenceof a naturally-occurring tribonectin is in the range of 220-280 kDa.Preferably, the apparent molecular weight of a tribonectin is less than230 kDa, more preferably less than 250 kDa, and most preferably lessthan 280 kDa. A protein or polypeptide fragment is defined as apolypeptide which has an amino acid sequence that is identical to part,but not all, of the amino acid sequence of a naturally-occurring proteinor polypeptide from which it is derived, e.g., MSF. The tribonectin maycontain a polypeptide, the amino acid sequence of which is at least 50%identical to the sequence of residues 200-1140, inclusive, of SEQ IDNO:1, e.g., it contains the amino acid sequence of residues 200-1140,inclusive, of SEQ ID NO:1, In another example, the polypeptide containsan amino acid sequence that is at least 50% identical to the sequence ofresidues 200-1167, inclusive, of SEQ ID NO:1, e.g., one having the aminoacid sequence identical to residues 200-1167, inclusive, of SEQ ID NO:1.The polypeptide contains an amino acid sequence that is at least 50%identical to the sequence of residues 200-1212, inclusive, of SEQ IDNO:1, e.g., the amino acid sequence of residues 200-1212, inclusive, ofSEQ ID NO:1, or the polypeptide contains an amino acid sequence that isat least 50% identical to the sequence of residues 200-1263, inclusive,of SEQ ID NO:1, e.g., an amino acid sequence identical to residues200-1263, inclusive, of SEQ ID NO:1. Preferably, the sequence of thepolypeptide lacks the amino acid sequence of residues 1-24, inclusive,of SEQ ID NO:1 and/or the amino acid sequence of residues 67-104,inclusive of SEQ ID NO:1.

The invention also features an isolated nucleic acid molecule encoding atribonectin. For example, the nucleic acid includes a sequence that isat least 50% identical to nucleotides 631-3453, inclusive, of SEQ IDNO:2. Preferably, the nucleic acid encodes a polypeptide with the aminoacid sequence of residues 200-1140 of SEQ ID NO:1. For example, thenucleic acid has a nucleotide sequence identical to that of nucleotides631-3453, inclusive, of SEQ ID NO:2, or a degenerate variant thereof. Anisolated nucleic acid molecule is a nucleic acid molecule that isseparated from the 5′ and 3′ coding sequences or non-coding sequenceswith which it is immediately contiguous in the naturally occurringgenome of an organism. Isolated nucleic acid molecules include nucleicacid molecules which are not naturally occurring, e.g., nucleic acidmolecules created by recombinant DNA techniques. For example, thenucleic acid of the invention includes nucleotides 631-3453, inclusive,of SEQ ID NO:2, but not nucleotides which are immediately contiguous tothose sequences in the naturally-occurring genomic sequence ornaturally-occurring cDNA.

Also within the invention is a method of lubricating a mammalian jointby contacting the joint with a tribonectin. The mammal is preferably ahuman, horse, dog, ox, donkey, mouse, rat, guinea pig, cow, sheep, pig,rabbit, monkey, or cat, and the joint is an articulating joint such as aknee, elbow, shoulder, hip, or any other weight-bearing joint.Tribonectins are administered intra-articularly. Therapeutic jointlubrication is also carried out by gene therapy, e.g., by contacting thejoint or synovial fluid with a nucleic acid encoding a tribonectin. Forexample, nucleic acids are administered to a synovial cavity byintra-articular injection.

In addition to functioning as a boundary lubricant in a mammalian joint,a tribonectin is used as a boundary lubricant between soft mammaliantissues such as skin or internal organs or between a mammalian tissueand a medical device such as a prosthetic implant. Accordingly, theinvention encompasses a biocompatible composition containing atribonectin in a form suitable for the inhibition of tissue adhesionformation. For example, the tribonectin is in the form of a film,membrane, foam, gel, or fiber. The term “film,” as used herein, means asubstance formed by compressing a foam or gel to a thin membrane, e.g.,by casting into a flat mold and air drying to a thin membrane, or bycompressing a gel or fibers, or by allowing or causing a gel or fibersto dehydrate. The term “foam,” as used herein, means a substance with aporous structure formed, e.g., by introduction of as air into atribonectin solution, suspension, gels, or fiber. The term“bioabsorbable,” as used herein, refers to the ability of atissue-compatible material to degrade in the body after implantation,into nontoxic products which are eliminated from the body ormetabolized. A “biocompatible” substance, as the term is used herein, isone that has no medically unacceptable toxic or injurious effects onbiological function. Tribonectin compositions for the prevention ofadhesions are also formulated as compositions suitable for extrusion,e.g., to form a mold upon which tissue can grow without adhering.

A method inhibiting adhesion formation between a first surface and asecond surface in a mammal is carried out by placing a tribonectinbetween the first and second surfaces in an amount sufficient to preventadhesion of the surfaces in the mammal. For example, one or both of thesurfaces is a mammalian tissue, and a tribonectin placed between themprevents formation of adhesions during the healing process.Alternatively the first or the second surface (or both) is an artificialdevice such as an orthopedic implant. Tissues to be treated includethose injured by surgical incision or trauma.

Also within the invention is a method for diagnosing osteoarthritis or apredisposition thereto by obtaining a biological sample from a mammaland measuring the amount of an MSF fragment in the biological sample. Anincrease in the amount compared to a control, e.g, a predetermined valueassociated with a negative diagnosis or a biological sample from amammal known to be free of osteoarthritis, indicates that the mammalsuffers from osteoarthritis or is predisposed to developingosteoarthritis. Any biological sample is suitable for testing in thediagnostic method; typically, the biological sample is synovial fluid,blood, serum, or urine. Preferably, the MSF fragment contains the aminoacid sequence of SEQ ID NO:3. Alternatively, the MSF fragment containsthe amino acid sequence of EPAPTT (SEQ ID NO:5; a product of trypsincleavage of a tribonectin) or the amino acid sequence of PTTKEP (SEQ IDNO:6; a product of elastase cleavage of a tribonectin).

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

DETAILED DESCRIPTION

A human lubricating polypeptide was purified from synovial fluid andfound to contain amino acids encoded by exons 2 and 4-12 of the MSF gene(but not exons 1 or 3). The gene encoding naturally-occurring fulllength MSF contains 12 exons, and the naturally-occurring MSF geneproduct contains 1404 amino acids with multiple polypeptide sequencehomologies to vitronectin including hemopexin-like and somatomedin-likeregions. Centrally-located exon 6 contains 940 residues. Exon 6 encodesa O-glycosylated mucin domain. A polypeptide encoded by nucleotides631-3453 of SEQ ID NO:2 provides boundary lubrication of articularcartilage.

TABLE 1 MSF amino acid sequenceMAWKTLPIYLLLLLSVFVIQQVSSQDLSSCAGRCGEGYSRDATCNCDYNCQHYMECCPDFKRVCTAELSCKGRCFESFERGRECDCDAQCKKYDKCCPDYESFCAEVHNPTSPPSSKKAPPPSGASQTIKSTTKRSPKPPNKKKTKKVIESEEITEEHSVSENQESSSSSSSSSSSSTIWKIKSSKNSAANRELQKKLKVKDNKKNRTKKKPTPKPPVVDEAGSGLDNGDFKVTTPDTSTTQHNKVSTSPKITTAKPINPRPSLPPNSDTSKETSLTVNKETTVETKETTTTNKQTSTDGKEKTTSAKETQSIEKTSAKDLAPTSKVLAKPTPKAETTTKGPALTTPKEPTPTTPKEPASTTPKEPTPTTIKSAPTTPKEPAPTTTKSAPTTPKEPAPTTTKEPAPTTPKEPAPTTTKEPAPTTTKSAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPTPTTPKEPAPTTKEPAPTTPKEPAPTAPKKPAPTTPKEPAPTTPKEPAPTTTKEPSPTTPKEPAPTTTKSAPTTTKEPAPTTTKSAPTTPKEPSPTTTKEPAPTTPKEPAPTTPKKPAPTTPKEPAPTTPKEPAPTTTKKPAPTAPKEPAPTTPKETAPTTPKKLTPTTPEKLAPTTPEKPAPTTPEELAPTTPEEPTPTTPEEPAPTTPKAAAPNTPKEPAPTTPKEPAPTTPKEPAPTTPKETAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKEPTSTTSDKPAPTTPKGTAPTTPKEPAPTTPKEPAPTTPKGTAPTTLKEPAPTTPKKPAPKELAPTTTKGPTSTTSDKPAPTTPKETAPTTPKEPAPTTPKKPAPTTPETPPPTTSEVSTPTTTKEPTTIHKSPDESTPELSAEPTPKALENSPKEPGVPTTKTPAATKPEMTTTAKDKTTERDLRTTPETTTAAPKMTKETATTTEKTTESKITATTTQVTSTTTQDTTPFKITTLKTTTLAPKVTTTKKTITTTEIMNKPEETAKPKDRATNSKATTPKPQKPTKAPKKPTSTKKPKTMPRVRKPKTTPTPRKMTSTMPELNPTSRIAEAMLQTTTRPNQTPNSKLVEVNPKSEDAGGAEGETPHMLLRPHVFMPEVTPDMDYLPRVPNQGIIINFMLSDETNICNGKPVDGLTTLRNGTLVAFRGHYFWMLSPFSPPSPARRITEVWGIPSPIDTVFTRCNCEGKTFFFKDSQYWRFTNDIKDAGYPKPIFKGFGGLTGQIVAALSTAKYKNWPESVYFFKRGGSIQQYIYKQEPVQKCPGRRPALNYPVYGEMTQVRRRRFERAIGPSQTHTIRIQYSPARLAYQDKGVLHNEVKVSILWRGLPNVVTSAISLPNIRKPDGYDYYAFSKDQYYNIDVPSRTARAITTRSGQTLSKVW YNCP (SEQ ID NO: 1)

TABLE 2 MSF cDNA    1 gcggccgcga ctattcggta cctgaaaaca acgatggcatggaaaacact tcccatttac   61 ctgttgttgc tgctgtctgt tttcgtgatt cagcaagtttcatctcaaga tttatcaagc  121 tgtgcaggga gatgtgggga agggtattct agagatgccacctgcaactg tgattataac  181 tgtcaacact acatggagtg ctgccctgat ttcaagagagtctgcactgc ggagctttcc  241 tgtaaaggcc gctgctttga gtccttcgag agagggagggagtgtgactg cgacgcccaa  301 tgtaagaagt atgacaagtg ctgtcccgat tatgagagtttctgtgcaga agtgcataat  361 cccacatcac caccatcttc aaagaaagca cctccaccttcaggagcatc tcaaaccatc  421 aaatcaacaa ccaaacgttc acccaaacca ccaaacaagaagaagactaa gaaagttata  481 gaatcagagg aaataacaga agaacattct gtttctgaaaatcaagagtc ctcctcctcc  541 tcctcctctt cctcttcttc ttcaacaatt tggaaaatcaagtcttccaa aaattcagct                                       EXON 6  601gctaatagag aattacagaa gaaactcaaa gtaaaagata acaagaagaa caaaactaaa  661aagaaaccta cccccaaacc accatttgta gattaagctt gaagttgatt ggacaatggt  721gacttcaagg tcacaactcc tgacacgtct accacccaac acaataaagt cagcacatct  781cccaagatca caacagcaaa accaataaat cccagaccca gtcttccacc taattctgat  841acatctaaag agacgtcttt gacagtgaat aaagagacaa cagttgaaac taaagaaact  901actacaacaa ataaacagac ttcaactgat ggaaaagaga agactacttc cgctaaagag  961acacaaagta tagagaaaac atctgctaaa gatttagcac ccacatctaa agtgctggct 1021aaacctacac ccaaagctga aactacaacc aaaggccctg ctctcaccac tcccaaggag 1081cccacgccca ccactcccaa ggagcctgca tctaccacac ccaaagagcc cacacctacc 1141accatcaagt ctgcacccac cacccccaag gagcctgcac ccaccaccac caagtctgca 1201cccaccactc ccaaggagcc tacacccacc accaccaagg agcctgcacc caccactccc 1261aaggagcctg cacccaccac caccaaggag cctgcaccca ccaccaccaa gtctgcaccc 1321accactccca aggagcctgc acccaccacc cccaagaagc ctgccccaac tacccccaag 1381gagcctgcac ccaccactcc caaggagcct acacccacca ctcccaagga gcctgcaccc 1441accaccaagg agcctgcacc caccactccc aaagagcctg cacccactgc ccccaagaag 1501cctgccccaa ctacccccaa ggagcctgca cccaccactc ccaaggagcc tgcacccacc 1561accaccaagg agccttcacc caccactecc aaggagcctg cacccaccac cactaagtct 1621gcacccacca ctaccaagga gcctgcaccc accactacca agtctgcacc caccactccc 1681aaggagcctt cacccaccac caccaaggag cctgcaccca ccactcccaa ggagcctgca 1741cccaccaccc ccaagaagcc tcccccaact acccccaagg agcctgcacc caccactccc 1801aaggaacctg cacccaccac caccaagaag cctgcaccca ccgctcccaa agagcctgcc 1861ccaactaccc ccaaggatac tggacccacc acccccaaga agctcacgcc caccaccccc 1921gagaagctcg cacccaccac ccctgagaag cccgcaccca ccacccctga ggagctcgca 1981cccaccaccc ctgaggagcc cacacccacc acccctgagg agcctgctcc caccactccc 2041aaggcagcgg ctcccaacac ccctaaggag cctgctccaa ctacccctaa ggagcctgct 2101ccaactaccc ctaaggagcc ttctccaact acccctaagq agactgctcc aactacccct 2161aaagggactg ctccaactac cctcaaggaa cctgcaccca ctactcccaa gaagcctgcc 2221cccaaggagc ttgcacccac cactaccaag gagcccacat ccaccacctc tgacaagccc 2281gctccaacta cccctaaggg gactgctcca actaccccta aggagcctgc tccaactacc 2341cctaaggagc ctgctccaac tacccctaag gggactgctc caactaccct caaggaacct 2401ccacccacta ctcccaagaa gcctgccccc aaggagcttg cacccaccac caccaagggg 2461cccacatcca ccacctctga caagcctgct ccaactacac ctaaggagac tgctccaact 2521acccccaagg agcctgcacc cactaccccc aagaagcctg ctccaactac tcctgagaca 2581cctcctccaa ccacttcaga ggtctctact ccaactacca ccaaggagcc taccactatc 2641cacaaaagcc ctgatgaatc aactcctgag ctttctgcag aacccacacc aaaagctctt 2701caaaacagtc ccaaggaacc tggtgtacct acaactaaga ctcctgcagc gactaaacct 2761gaaatgacta caacagctaa agacaagaca acagaaagag acttacgtac tacacctgaa 2921actacaactg ctgcacctaa gatgacaaaa gagacagcaa ctacaacaga aaaaactacc 2881gaatccaaaa taacagctac aaccacacaa gtaacatcta ccacaactca agataccaca 2941ccattcaaaa ttactactct taaaacaact actcttgcac ccaaagtaac tacaacaaaa 3001aagacaatta ctaccactga gattatgaac aaacctgaag aaacagctaa accaaaagac 3061agagctacta attctaaagc gacaactcct aaacctcaaa agccaaccaa agcacccaaa 3121aaacccactt ctaccaaaaa gccaaaaaca atgcctagag tgagaaaacc aaagacgaca 3181ccaactcccc gcaagatgac atcaacaatg ccagaattga accctacctc aagaatagca 3241gaagccatgc tccaaaccac caccagacct aaccaaactc caaactccaa actagttgaa 3301gtaaatccaa agagtgaaga tgcaggtggt gctgaaggag aaacacctca tatgcttctc 3361aggccccatg tgttcatgcc tgaagttact cccgacatgg attactcacc gagagtaccc 3421aatcaaggca ttatcatcaa tcccatgctt tccgatgaga ccaatatatg caatggtaag 3481ccagtagatg gactgactac tttgcgcaat gggacattag ttgcattccg aggtcattat 3541ttctggatgc taagtccatt cagtccacca tctccagctc gcagaattac tgaagtttgg 3601ggtattcctt cccccattga tactgttttt actaggtgca actgtgaagg aaaaactttc 3661ttctttaagg attctcatta ctggcgtttt accaatgata taaaagatgc agggtacccc 3721aaaccaattt tcaaaggatt tggaggacta actggacaaa tagtggcagc gctttcaaca 3781gctaaataca agaactggcc tgaatctgtg tattttttca agagaggegg cagcattcag 3841cagtatattt ataaacagga acctgtacag aagtgccctg gaagaaggcc tgctctaaat 3901tatccagtgt atggagaaat gacacaggtt aggagacgtc gctttgaacg tgctatagga 3961ccttctcaaa cacacaccat cagaattcaa tattcacctg ccagactggc ttatcaagac 4021aaaggtgtcc ttcataatga agttaaagtg agtatactgt ggagaggact tccaaatgtg 4081tttacctcag ctatatcact tcccaacatc agaaaacctg acggctatga ttactatgcc 4141ttttctaaag atcaatacta taacattgat gtgcctagta gaacagcaag agcaattact 4201actcgttctg ggcagacctt atccaaagtc tggtacaact gtccttagac tgatgagcaa 4262aggaggagtc aactaatgaa gaaatgaata ataaattttg acactgaaaa acattttatt 4321aataaagaat attgacatga gtataccagt ttatatataa aaatgttttt aaacttgaca 4381atcattacac taaaacagat ttgataatct tattcacage tgttattgtt tacagaccat 4441ttaattaata tttcctctgt ttattcctcc tctccctccc attgcatggc tcacacctgt 4501aaaagaaaaa agaatcaaat tgaatatatc ttttaagaat tcaaaactag tgtattcact 4561taccctagtt cattataaaa aatatctagg cattgtggat ataaaactgt tgggtattct 4621acaacttcaa tggaaattat tacaagcaga ttaatccctc tttttgtgac actagtacaa 4681tctaaaagtt atattggaaa acatggaaat attaaaattt tacactttta ctagctaaaa 4741cataatcaca aagctttatc gtgttgtata aaaaaattaa caatataatg gcaataggta 4801gagatacaac aaatgaatat aacactataa cacttcatat tttccaaatc ttaatttgga 4861tttaaggaag aaatcaataa atataaaata taagcacata tttattatat atctaaggta 4921tacaaatctg tctacatgaa gtttacagat tggtaaatat cacctgctca acatgtaatt 4981atttaataaa actttggaac attaaaaaaa taaattggag gcttaaaaaa aaaaaaaaaa 5041 a(SEQ ID NO: 2)

TABLE 3 MSF Exon Boundaries Amino acid sequence Nucleotide sequence inExon in SEQ ID NO: 1 SEQ ID NO: 2 1 1-24, inclusive 34-105, inclusive 225-66, inclusive 106-231, inclusive 3 67-104, inclusive 232-345,inclusive 4 105-155, inclusive 346-498, inclusive 5 156-199, inclusive499-630, inclusive 6 200-1140, inclusive 631-3453, inclusive 71141-1167, inclusive 3454-3534, inclusive 8 1168-1212, inclusive3535-3670, inclusive 9 1213-1263, inclusive 3671-3822, inclusive 101264-1331, inclusive 3823-4026, inclusive 11 1332-1371, inclusive4027-4146, inclusive 12 1372-1404, inclusive 4147-4245, inclusive

The boundary lubricant isolated from synovial fluid is analternatively-spliced variant of MSF. This alternatively-spliced variantwas found to be the composition present in synovial fluid that conferslubricating capabilities to the articular joint. The boundary lubricantisolated from human synovial fluid contains amino acids encoded by exons2, and 4-12 of the MSF gene, i.e., the alternative splice variant lacksamino acids encoded by exons 1 and 3 of the MSF gene. A recombinant orchemically-produced polypeptide containing at least exon 6 (but notexons 1 or 3) of MSF is useful to prevent and/or treat osteoarthriticdisease. A recombinant or chemically-produced lubricating polypeptidecontaining at least one repeat of the amino acid sequence KEPAPTT (SEQID NO:3) either identically or with conservative substitution is alsoadministered to lubricate mammalian joints.

Production and Purification of Recombinant Lubricating Polypeptides

To produce recombinant polypeptides, DNA containing exon 6 of MSF(nucleotides 631-3453 of SEQ ID NO:2) in an appropriate expressionvector is transfected into a cell. The DNA can also contain some or allof exon 7 (nucleotides 354-3534 of SEQ ID NO:2), exon 8 (nucleotides3535-3670 of SEQ ID NO:2), or exon 9 (nucleotides 3671-3822 of SEQ IDNO:2) of the MSF gene. Primers for polymerase chain reaction (PCR)methods to generate DNA which spans various exons of MSF are shownbelow.

TABLE 4 PCR Primers MSF exons Forward Primer Reverse Primer exon 25′AGATTTATCAAGCTGT 5′TTTACAGGAAAGC GCAGGGAG3′ TCCGCAGTG3′ (SEQ ID NO: 7)(SEQ ID NO: 8) exon 6 5′TCAAGGTCACAACTCC 5′CTCTCGGTAAGTA TGACACG3′ATCCATGTCGG3′ (SEQ ID NO: 9) (SEQ ID NO: 10) exons 2-125′TTGTTGCTGCTGTCTG 5′TGGATAAGGTCTG TTTTCG3′ CCCAGAACGAG3′ (SEQ ID NO:11) (SEQ ID NO: 12) exons 6-12 5′TCAAGGTCACAACTCC 5′GATGGTGTGTGTTTGACACG3′ TGAGAAGGTCC3′ (SEQ ID NO: 13) (SEQ ID NO: 14)Methods of designing forward and reverse primers used to make DNAs whichencode tribonectin polypeptides of varying lengths and which incorporatevarious exons of the MSF gene, e.g., to make polypeptide encoded byexons 2, 4-12; exons 6-9; and exons 2, 4-9, are well known in the art ofmolecular biology. Standard methods for transfecting cells with isolatednucleic acid are well known to those skilled in the art of molecularbiology. For example, prokaryotic or eukaryotic cells in culture aretransfected with the DNA of the invention operatively linked toexpression control sequences appropriate for high-level expression inthe cell. Such cells are useful for producing large amounts of thelubricating polypeptide, which are purified using standard methods. Thelubricating polypeptides are used therapeutically for treatment orprevention of arthritic diseases. The polypeptides are also used toraise antibodies against a naturally-occurring or recombinantly-producedlubricating glycoproteins or glycopeptides.

For example, the recombinant gene product is expressed as a fusionprotein and purified using a commercially available expression andpurification system, e.g., the pFLAG expression system (IBI). Theexpression systems that may be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA, or cosmid DNA expression vectors containing the nucleicacid molecules described herein. For production of glycosylatedpolypeptides, eukaryotic expression systems are used. Yeast (forexample, Saccharomyces and Pichia) transformed with recombinant yeastexpression vectors containing the recombinant nucleic acid encoding atribonectin polypeptide are used. Insect cell systems infected withrecombinant virus expression vectors (for example, baculovirus)containing the nucleic acid molecules encoding a tribonectin andmammalian cell systems (e.g., COS, CHO, BHK, 293, VERO, HeLa, MDCK,WI38, and NIH 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,the metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter and the vaccinia virus 7.5K promoter) are alsouseful. In addition to clinical applications, recombinant polypeptidesare injected into a rabbit or rodent to produce antibodies as describedbelow.

The synovial fluid of an inflamed or injured joint contains proteolyticenzymes that degrade lubricating proteins or polypeptides. For example,infiltrating immune cells such as neutrophils secrete trypsin and/orelastase. Even a minor injury to an articulating joint or aninflammatory state can result in cellular infiltration and proteolyticenzyme secretion resulting in traumatic synovitis. Synovitis for aperiod of a few days or weeks can result in the loss of thecytoprotective layer of a joint, which in turn leads to the loss ofcartilage. Non-lubricated cartilaginous bearings may experiencepremature wear which may initiate osteoarthritis. Individuals whoclinically present with a traumatic effusion (e.g., “water on the knee”)are predisposed to developing osteoarthritis; the elaboration ofproteolytic enzymes degrades and depletes naturally-occurringlubricating compositions in the synovial fluid. Depletion of naturallubricating compositions occurs in other inflammatory joint diseasessuch as rheumatoid arthritis. Replacing or supplementing the synovialfluid of such injured joints with the lubricating compositions of theinvention prevents the development of osteoarthritis in the long term(e.g., years, even decades later) and immediately lubricates the jointto minimize short term damage.

Analogs, homologs, or mimetics of lubricating peptides which are lesssusceptible to degradation in vivo are used to lubricate mammalianjoints. Analogs can differ from the naturally-occurring peptides byamino acid sequence, or by modifications which do not affect thesequence, or both. Modifications (which do not normally alter primarysequence) include in vivo or in vitro chemical derivitization ofpolypeptides, e.g., acetylation or carboxylation. Also included aremodifications of glycosylation, e.g., those made by modifying theglycosylation patterns of a polypeptide during its synthesis andprocessing or in further processing steps, e.g., by exposing thepolypeptide to enzymes which affect glycosylation, e.g., mammalianglycosylating or deglycosylating enzymes.

Where proteolytic degradation of the peptides following injection intothe subject is a problem, replacement of a particularly sensitivepeptide bond with a noncleavable peptide mimetic bond renders theresulting peptide more stable, and thus more useful as a therapeutic. Torender the therapeutic peptides less susceptible to cleavage bypeptidases such as trypsin or elastase, the peptide bonds of a peptidemay be replaced with an alternative type of covalent bond (a “peptidemimetic”). Trypsin, elastase, and other enzymes may be elaborated byinfiltrating immune cells during joint inflammation. Trypsin cleaves apolypeptide bond on the carboxy-side of lysine and arginine; elastasecleaves on the carboxy-side of alanine, glycine. Thrombin, a serineprotease which is present in hemorrhagic joints, cleaves a peptide bondon the carboxy-side of arginine. Collagenases are a family of enzymesproduced by fibroblasts and chondrocytes when synovial metabolism isaltered (e.g., during injury). These enzymes cut on the carboxy-side ofglycine and proline. One or more peptidase-susceptible peptide bonds,e.g, those which appear in the KEPAPTT (SEQ ID NO:3) repeat sequence,are altered (e.g., replaced with a non-peptide bond) to make the siteless susceptible to cleavage, thus increasing the clinical half-life ofthe therapeutic formulation.

Such mimetics, and methods of incorporating them into polypeptides, arewell known in the art. Similarly, the replacement of an L-amino acidresidue with a D-amino acid is a standard way of rendering thepolypeptide less sensitive to proteolysis. Also useful areamino-terminal blocking groups such as t-butyloxycarbonyl, acetyl,theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl,methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl.

Clinical formulations of tribonectins may also contain peptidaseinhibitors such as N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethylketone(an inhibitor of elastase). Other clinically acceptable proteaseinhibitors (e.g., as described in Berling et al., 1998, Int. J.Pancreatology 24:9-17) such as leupeptin, aprotinin, α1-antitrypsin,α2-macroglobulin, α1-protease inhibitor, antichymotrypsin (ACHY),secretory leukocyte protease inhibitor (PSTI) are also co-administeredwith a tribonectin to reduce proteolytic cleavage and increase clinicalhalflife. A cocktail of two or more protease inhibitors can also becoadministered.

Compositions of tribonectin polypeptides or nucleic acids encoding thepolypeptides are formulated in standard physiologically-compatibleexcipients known in the art., e.g., phosphate-buffered saline (PBS).Other formulations and methods for making such formulations are wellknown and can be found in, e.g., “Remington's Pharmaceutical Sciences”.Tribonectins are also formulated with non-crosslinked hyaluronic acidpreparations or viscosupplementation compositions, such as cross-linkedhyaluronic acid preparations. When a tribonectin is added to aviscosupplement formulation, the interaction of the tribonectin withhyaluronic acid reduces the viscosity of the viscosupplement.

Methods of making a glycopeptide and determining % glycosylation areknown in the art, e.g., as described in U.S. Pat. No. 5,767,254. Thepresence of N-acetylgalactosamine is indicative of the presence ofO-linked oligosaccharides (or Ser/Thr-linked oligosaccharides) in whichGalNAc is commonly found in O-glycosidic alpha-linkage directly to aminoacid. The presence of O-linked oligosaccharide is also detected bybinding to Jacalin-Sepharose, an immobilized plant lectin that binds tothe core disaccharide sequence Gal β(1-3)GalNAc linked to Ser/Thr inglycoproteins, or peanut agglutinin, which binds to β(1-3)Gal-GalNAC.O-linked oligosaccharides are distinguished from N-linkedoligosaccharides using standard methods. For example, oligosaccharidesin O-glycosidic linkage, but not in N-glycosidic linkage, aresusceptible to release from peptide by treatment with mild base in thepresence of sodium borohydride (50 mM NaOH, 1M NaBH₄, 16 hr at 45° C.)to cause a beta-elimination reaction.

Veterinary Applications

Canine osteoarthritis is a prevalent clinical disorder that is treatedusing the methods described herein. Osteoarthritis afflicts an estimatedone in five adult dogs; an estimated 8 million dogs suffer from thisdegenerative, potentially debilitating disease. Yet, many owners do notrecognize the signs of chronic canine pain. While any dog can developosteoarthritis, those most at risk are large breeds, geriatric dogs,very active dogs (such as working or sporting animals), and those withinherited joint abnormalities such as hip or elbow dysplasia.

Equine degenerative joint disease such as osteoarthritis is a cause oflameness and impaired performance in horses. As with humans and othermammals, degenerative joint diseases which affect horses are progressivedisorders of synovial joints characterized by articular cartilagedegeneration and joint effusion. Acute or chronic trauma, overuse,developmental disease, joint instability and old age leads to synovitis,impaired chondrocyte metabolism, and the formation of fissures in thejoint cartilage. Destructive enzymes such as trypsin, elastase,stromelysin and hyaluronidase are released into the joint where theydegrade synovial fluid and cartilage components, resulting in decreasedsynovial fluid viscosity, poor lubrication, depressed cartilagemetabolism and enhanced wear resulting in pain and cartilage erosion.Current therapeutic approaches include medications for pain relief andanti-inflammatory drugs. The compositions and methods described hereinare useful to replenish the lubricating capabilities of the affectedjoint.

Administration of Therapeutic Polypeptides

Standard methods for delivery of peptides are used. Such methods arewell known to those of ordinary skill in the art. For intra-articularadministration, tribonectin is delivered to the synovial cavity at aconcentration in the range of 20-500 μg/ml in a volume of approximately0.1-2 ml per injection. For example, 1 ml of a tribonectin at aconcentration of 250 μg/ml is injected into a knee joint using a fine(e.g., 14-22 gauge, preferably 18-22 gauge) needle. The compositions ofthe invention are also useful for parenteral administration, such asintravenous, subcutaneous, intramuscular, and intraperitoneal.

For prevention of surgical adhesions, the tribonectins described hereinare administered in the form of gel, foam, fiber or fabric. Atribonectin formulated in such a manner is placed over and betweendamaged or exposed tissue interfaces in order to prevent adhesionformation between apposing surfaces. To be effective, the gel or filmmust remain in place and prevent tissue contact for a long enough timeso that when the gel finally disperses and the tissues do come intocontact, they will no longer have a tendency to adhere. Tribonectinsformulated for inhibition or prevention of adhesion formation (e.g, inthe form of a membrane, fabric, foam, or gel) are evaluated forprevention of post-surgical adhesions in a rat cecal abrasion model(Goldberg et al., In Gynecologic Surgery and Adhesion Prevention.Willey-Liss, pp. 191-204, 1993). Compositions are placed aroundsurgically abraded rat ceca, and compared to non-treated controls(animals whose ceca were abraded but did not receive any treatment). Areduction in the amount of adhesion formation in the rat model in thepresence of the tribonectin formulation compared to the amount in theabsence of the formulation indicates that the formulation is clinicallyeffective to reduce tissue adhesion formation.

Tribonectins are also used to coat artificial limbs and joints prior toimplantation into a mammal. For example, such devices are dipped orbathed in a solution of a tribonectin, e.g, as described in U.S. Pat.Nos. 5,709,020 or 5,702,456.

Lubricating polypeptides are at least about 10 amino acids (containingat least one KEPAPTT (SEQ ID NO:3)) or XXTTTX (SEQ ID NO:4) repeat),usually about 20 contiguous amino acids, preferably at least 40contiguous amino acids, more preferably at least 50 contiguous aminoacids, and most preferably at least about 60 to 80 contiguous aminoacids in length. For example, the polypeptide is approximately 500 aminoacids in length and contains 76 repeats of KEPAPTT (SEQ ID NO:3). Thepolypeptide is less than 1404 residues in length, e.g., it has the aminoacid sequence of naturally-occurring MSF (SEQ ID NO:1) but lacks atleast 5, 10, 15, 20, or 24 amino acids at the N-terminus ofnaturally-occurring MSF. Such peptides are generated by methods known tothose skilled in the art, including proteolytic cleavage of arecombinant MSF protein, de novo synthesis, or genetic engineering,e.g., cloning and expression of at least exon 6, 7, 8, and/or 9 of theMSF gene.

Tribonectin polypeptides are also biochemically purified. The enzymechymotrypsin cleaves at sites which bracket amino acids encoded by exon6 of the MSF gene.

Thus, a polypeptide containing amino acids encoded by exon 6 of the MSFgene (but not any other MSF exons) is prepared from anaturally-occurring or recombinantly produced MSF gene product byenzymatic digestion with chymotrypsin. The polypeptide is then subjectedto standard biochemical purification methods to yield a substantiallypure polypeptide suitable for therapeutic administration, evaluation oflubricating activity, or antibody production.

Therapeutic compositions are administered in a pharmaceuticallyacceptable carrier (e.g., physiological saline). Carriers are selectedon the basis of mode and route of administration and standardpharmaceutical practice. A therapeutically effective amount of atherapeutic composition (e.g., lubricating polypeptide) is an amountwhich is capable of producing a medically desirable result, e.g.,boundary lubrication of a mammalian joint, in a treated animal. Amedically desirable result is a reduction in pain (measured, e.g., usinga visual analog pain scale described in Peyron et al., 1993, J.Rheumatol. 20 (suppl. 39):10-15) or increased ability to move the joint(measured, e.g., using pedometry as described in Belcher et al., 1997,J. Orthop. Trauma 11:106-109). Another method to measure lubricity ofsynovial fluid after treatment is to reaspirate a small volume ofsynovial fluid from the affected joint and test the lubricatingproperties in vitro using a friction apparatus as described herein.

As is well known in the medical arts, dosage for any one animal dependson many factors, including the animal's size, body surface area, age,the particular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. Administration is generally local to an injured orinflamed joint. Alternatively, the polypeptides are administered via atimed-release implant placed in close proximity to a joint for slowrelease at the site of an injured or inflamed joint.

Gene Therapy

Gene therapy is carried out by administering to a mammal a nucleic acidencoding a therapeutic lubricating polypeptide, e.g., DNA encoding oneor more repeats or the amino acid sequence KEPAPTT (SEQ ID NO:3) or DNAencoding a lubricating fragment of MSF, by standard vectors and/or genedelivery systems. Suitable gene delivery systems include liposomes,receptor-mediated delivery systems, naked DNA, and viral vectors such asherpes viruses, retroviruses, adenoviruses and adeno-associated viruses.

In addition to a gene delivery system as described above, thetherapeutic composition may include a pharmaceutically acceptablecarrier, e.g., a biologically compatible vehicle such as physiologicalsaline, suitable for administration to an animal. A therapeuticallyeffective amount of a nucleic acid or polypeptide composition is anamount which is capable of producing a medically desirable result in atreated animal, e.g., a reduction in pain associated with jointmovement, an increase in lubricating function of synovial fluid.

Parenteral administration, such as intravenous, subcutaneous,intramuscular, and intraperitoneal delivery routes, may be used todeliver the compound. Preferably, therapeutic compositions such asnucleic acids or polypeptides are delivered intra-articularly. Dosagefor any one patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs (e.g., anti-inflammatory drugs, viscotherapeutic drugs)being administered concurrently. A preferred dosage for administrationof nucleic acids is from approximately 10⁶ to 10²² copies of the nucleicacid molecule.

DNA is be introduced into target cells of the patient by standardvectors, e.g., a vector which contains DNA encoding a tribonectinoperably linked to a promoter sequence. Suitable gene delivery systemsmay include liposomes, receptor-mediated delivery systems, naked DNA,and viral vectors such as herpes viruses, retroviruses, andadenoviruses, among others.

DNA may be administered locally using an adenovirus or adeno-associatevirus delivery system using standard methods. For example, methods ofdelivering DNA intra-articularly to synovial fluid and methods ofdelivering DNA to cells from synovial fluid (e.g, synovial fibroblastsor chondrocytes) are described in U.S. Pat. No. 5,858,355. The onlycis-acting sequences required for replication and packaging ofrecombinant adeno-associated virus (AAV) vector are the AAV terminalrepeats. Up to 4 kb of DNA is inserted between the terminal repeatswithout effecting viral replication or packaging. To package arecombinant AAV vector, a plasmid containing the terminal repeats andDNA encoding a therapeutic polypeptide is co-transfected into cells witha plasmid that expresses AAV rep and capsid proteins. The transfectedcells are then infected with adeno-associated virus, and recombinant AAVvirus containing the desired sequences is isolated from cellsapproximately 48-72 hours after transfection. Recombinant virus is thenadministered for gene therapy applications using known methods.

Electroporation is another method of introducing DNA into target cells,e.g., synovial fibroblasts or chondrocytes, ex vivo. Cells to beelectroporated are placed into Hepes buffer saline (HBS) at aconcentration of about 10⁷ cells per ml. The DNA to be electroporated isadded at a concentration of approximately 5-20 μg/ml of HBS. The mixtureis placed into an electroporation device and an electric field isapplied according to standard protocols, e.g., in a range of betweenabout 250 and 300 volts. Following introduction of DNA into synovialcells ex vivo, the genetically modified autologous synovial cells aretransplanted back into the donor by intra-articular injection.Approximately 10⁷ cells are injected intra-articularly into joints in avolume of approximately 1 ml.

Synovial cells into which DNA is introduced are obtained using routinemethods, e.g., through an arthroscope. The arthroscope is a small,hollow rod inserted into the knee via a small puncture wound whichallows access to a surgical instrument to recover synovial cellsarthroscopically. In some cases, the synovial cells in arthroscopicallyexcised tissue are aseptically recovered by enzymatic digestion of theconnective tissue matrix. For example, the synovium is cut into piecesof approximately 1 mm diameter and digested sequentially with trypsin(0.2% w/v in Gey's Balanced Salt Solution) for 30 minutes at 37° C., andcollagenase (0.2% w/v in Gey's Balanced Salt Solution) for 2 hours at37° C. A suspension of genetically-modified cells is injected into arecipient mammalian joint. Intra-articular injections of this type areroutine and carried out in the doctor's office without additionalsurgical intervention. Repeat injections are carried out as needed.

Alternatively, the DNA (naked or packaged in a virus) is formulated in asuitable pharmaceutical carrier and injected intra-articularly. Genetherapy is also administered as a prophylactic measure to prevent thedevelopment of osteoarthritis in those individuals determined to behighly susceptible of developing this disease, e.g., those who havesuffered an acute joint injury. Direct intra-articular injection of aDNA encoding a therapeutic polypeptide into a joint results intransfection of the recipient synovial cells to allow expression of DNA.

Drugs which stimulate an endogenous tribonectin promoter, e.g., TGFβ,may also be administered as described above to increase the level ofsynovial expression.

Production of Antibodies Specific for Synovial Lubricating Polypeptides

Antibodies specific for lubricating polypeptides are obtained bytechniques well known in the art. Such antibodies can be polyclonal ormonoclonal. Polyclonal antibodies can be obtained, for example, by themethods described in Ghose et al., Methods in Enzymology, Vol. 93,326-327, 1983. For example, a lubricating polypeptide encoded bynucleotides 632-3453 of SEQ ID NO:2 is used as an immunogen to stimulatethe production of polyclonal antibodies in the antisera of a rabbit.Similar methods can be used to raise antisera in animals such as goats,sheep, and rodents.

Monoclonal antibodies are obtained by the well known process describedby Milstein and Kohler in Nature, 256:495-497, 1975, or as modified byGerhard, Monoclonal Antibodies, Plenum Press, 1980, pages 370-371.Hybridomas are screened to identify those producing antibodies that arehighly specific for a synovial lubricating polypeptide. Preferably, theantibody has an affinity of at least about 10⁸ liters/mole and morepreferably, an affinity of at least about 10⁹ liters/mole. Monoclonal orpolyclonal antibodies provide a means of rapidly purifying largequantities of recombinant lubricating polypeptides.

In addition to antibodies which are directed to the peptide core of atribonectin, an antibody directed to a sugar portion or to aglycopeptide complex of a tribonectin is desirable. To generate anantibody to the peptide core, a peptide spanning amino acids 200-350 ofSEQ ID NO:1 is used. Shorter peptides, e.g., 8-15 amino acids in length,which are identical to an 8-15 amino acid portion of amino acids 200-350of SEQ ID NO:1 are also used to generate such antibodies. Other peptidesto be used as immunogens for antibodies specific for the peptide core ofa tribonectin include those which are in the region of amino acids 24-66of SEQ ID NO:1, amino acids 105-155 of SEQ ID NO:1, or amino acids156-199 of SEQ ID NO:1. To generate antibodies which bind to aglycosylated tribonectin polypeptide (but not a deglycosylated ornonglycosylated form), the immunogen is preferably a glycopeptide, theamino acid sequence of which spans a highly glycosylated portion of atribonectin, e.g, a peptide with an amino acid sequence of residues200-1140 of SEQ ID NO:1. Shorter glycopeptides, e.g., 8-15 amino acidsin length, within the same highly glycosylated region are also used asimmunogens. Methods of generating antibodies to highly glycosylatedbiomolecules are known in the art, e.g., as described by Schneerson etal., 1980, J. Exp. Med. 152:361-376.

Methods of Diagnosis

Osteoarthritis is a disease that develops slowly and is difficult todiagnose until its late stages when joint pain often compels anindividual to seek medical treatment. Early diagnosis of osteoarthritisor a predisposition to develop the disease allows early intervention toprevent or reduce the development of advanced osteoarthritis. Theinvention provides methods of early detection of this disease or apredisposition to develop it by testing bodily fluids such as serum orurine for the presence of fragments of naturally-occurring tribonectinsor the presence of fragments of MSF. Detection and quantitation of suchpeptides in biological fluids is well known in the art. For example, astandard sandwich ELISA assay is carried out using two differentantibodies (e.g., a first antibody which binds to an oligosaccharideportion of the glycopeptide and a second antibody which binds to thepeptide core of the glycopeptide) to a naturally-occurring tribonectin.Alternatively, standard protein sequencing by liquid chromatography andmass spectroscopy, as is described below, is used to detect MSFfragments in biological samples. A control value is a predeterminedvalue associated with a negative diagnosis; alternatively, a controlsample is a biological sample from a mammal known to be free ofosteoarthritis. An increase in the amount compared to a control value orsample indicates that the mammal suffers from osteoarthritis or ispredisposed to developing osteoarthritis.

Characterization of a Tribonectin from Human Synovial Fluid

Aliquots of synovial fluid from patients undergoing diagnosticarthroscopy and total knee replacement were collected and assayed in thefriction apparatus. In both cases, the synovial fluid was aspiratedprior to initiation of any surgical procedure and immediatelycentrifuged at 10,000×g at 4° C. for 2 hrs to remove cellular debris.Samples which were contaminated with blood were discarded. Aliquots withnormal lubricating ability were pooled and stored at −20° C.

Purification and Isolation of a Tribonectin

Human synovial fluid (200 ml) was filtered through 0.22 μm sterilefilter units (Nalgene) at 4° C. over two days. Retentate was scraped offfilter membranes and resuspended with 50 mM NaAc buffer, pH 5.5, to theoriginal synovial fluid volume containing proteolytic inhibitors: 1 mMphenylmethyl sulfonyl fluoride (PMSF), 1 mM parachloromercuricbenzoicacid (PCMB), and 10 mM ethylenediamine tetraacetate (EDTA). Digestion ofhyaluronic acid was carried out at 37° C. by streptomyces hyaluronidaseat 1 U/ml of resuspended synovial fluid. The digest was loaded on a DEAEcolumn (Whatman International, Maidstone, UK) settled volume of 300 ml,equilibrated with NaAc buffer, 50 mM and washed with 1.5 L of the samebuffer. The material with lubricating activity was eluted off of theDEAE matrix with 1 M NaCl. A 1 L wash was collected and concentrated viaa 500 ml Amicon flow cell with an XM-100 membrane (mw cutoff 100 kDa).The concentrated sample was dialyzed against 25 mM phosphate buffer, pH7.4, containing 0.15 M NaCl and 0.5 mM CaCl₂.

The DEAE-bound concentrate was loaded onto a peanut agglutinin(PNA)-agarose affinity column with a settled bed volume of 25 ml,equilibrated at room temperature with 25 mM phosphate and 0.15 NaClbuffer, pH 7.4. Unbound protein was eluted with the same buffer untilabsorbance at 230 and 280 nm decreased to background. Material withlubricating activity was maximally eluted in the presence of a step-wisegradient of α-lactose at a concentration of 0.07 M in 25 mM Tris and0.15 M NaCl at pH 7.4. This material was loaded onto an Actigel ALDagarose (Sterogene Bioseparations, Arcadia, Calif.) coupled via aminegroups to a murine monoclonal antibody against human fibronectin (ZymedLaboratories Inc., San Francisco, Calif.) to remove fibronectin as acontaminant. Eluted material was assayed for purity on SDS-PAGE (5-15%acrylamide) stained with Coomassie blue and by HPLC.

Protein electrophoresis standards were from GibcoBRL (Grand Island,N.Y.), and DNA ladder standard was from FMC Bioproducts (Rockland, Me.).

High Pressure Liquid Chromatography

A μBondpak C18 3.9×150 mm column (Waters, Milford, Mass.) was eluted inreverse phase with 45% (v/v) methanol (Sigma) and 5% (v/v) acetonitrile(Aldrich) HPLC grade at 1 ml/min at 35° C. The eluate was assayed by aphoto diode array detector PDA 996 (Waters), and material in peakfractions were analyzed by purity plots calculated using Millenium 32software (Waters).

Friction Apparatus

A standard friction apparatus (e.g., an apparatus described by Jay etal., 1992, Conn. Tiss. Res. 28:71-88 or Jay et al., 1998, J. Biomed.Mater. Res. 40:414-418). Natural latex was oscillated against a ring ofpolished glass with a constant contact area of 1.59 cm². The bearingsystem was axially loaded within a gimbals system free to rotate aroundtwo perpendicular horizontal axes. Latex and glass as bearing materialswere chosen because they offer a flat surface with small asperityheights on the order of 0.05 mm. Latex, like cartilage, is compliant.Within the gimbals system, these surfaces possess near perfectco-planarity. Accordingly, fluid wedges were not generated and only athin layer of boundary fluid was present. The entraining velocity (i.e.,sliding speed) was 0.37 mm/sec with a constant contact pressure of0.35×10⁶ N/m².

The friction apparatus recorded displacements of the gimbals systemaround the vertical loading axis through a linear displacement voltagetransducer, the output voltage of which was directly proportional to themagnitude of the frictional torque. The peak to peak amplitude of thissignal was related to μ by a previous calibration with known frictionaltorque.

Test surfaces were cleaned extensively before use. A 3.8×3.8 cm piece oflatex strapped onto the stainless steel stud was washed under runningdistilled deionized water (DDW) for 2 min. It was then placed in ashallow bath of 0.9% NaCl physiological saline (PS). The glass slide wasscrubbed with a 1% (v/v) 7× detergent (Flow Laboratories, McLean, Va.)solution in DDW for 10 min and then allowed to soak in the same solutionat 100° C. A 5 min. scrubbing was also performed with the hot 7×solution followed by rinsing for 2-4 min. under running DDW.

The μ was measured at 35° C. and was preceded by a baseline measurementof the μ with PS. Lubrication was manifested by a reduction of μrelative to the μ of PS. Negative delta μ values indicate lubrication,whereas positive values indicate friction. Addition of 200 μl of PS andlater 200 μl of test lubricant was followed by bringing the bearingsurfaces close enough so that the solution wet both surfaces. After 5min for equilibration, the latex-coated bearing was brought to rest onthe glass as it was oscillating. Peak to peak voltages wereautomatically recorded after 1, 3 and 5 mins. At this point, thesurfaces were separated for 2 min. and then brought back together foranother 5 min session. The 3 and 5 min. μ values of the last two 5 min.sessions typically stabilized and were recorded.

Human serum fibronectin was purchased from Sigma Chemical (F0895, St.Louis, Mo.) and dialyzed against PS before use in the frictionapparatus.

Boundary lubricants exert their effect by changing the physico-chemicalcharacteristics of a surface. Bearing surfaces must generate a mutualrepulsion in order to be lubricated in the boundary mode. Typical roomtemperature examples of boundary lubricants are graphite, teflon andmolybdenum sulphide. Such compositions reduce friction between bearingsurfaces, and therefore, are used as positive controls in assay tomeasure the lubricating properties of tribonectins. Tribonectins areboundary lubricants that can have an amphipathic character by coatingnon-biologic hydrophobic surfaces such as latex. The oligosaccharidecomponent of a tribonectin networks with the surrounding aqueousenvironment. When the ultimate and penultimate sugars are removed from anaturally-occurring tribonectin purified from synovial fluid, thelubricating ability is eliminated.

The latex:glass arthrotripsometer offers an expedient way to testpurified biological lubricating factors repetitively withreproducibility. Natural latex and polished glass represent bearingsurfaces with little if any variation in physico-chemical characteristicfrom test to test. By contrast, resected cartilage apposed to eitherpolished glass or cartilage itself will experience deformation thatcannot be accurately controlled. The μ observed in a cartilage-cartilagebearing lubricated by synovial fluid was between 0.005 and 0.024. Thevalues of μ in the latex:glass system were appreciably higher andtypically 0.04 or less. Differences in μ between the bearing materialsare attributed to the 80% (w/w) water content of cartilage.

Protein Sequencing by Liquid Chromatography and Mass Spectrometry (LCMS)

Standard LCMS was carried out on tryptic digests of the purifiedlubricating material described above. Excised bands from 2 mm thick5-20% gradient SDS-PAGE gels (Bio-rad Laboratories, Hercules, Calif.)containing the lubricating material was analyzed. The material wasdeglycosylated by NaNase III and O-glycosidase DS (Glyko, Novato,Calif.). Deglycosylation was carried out with the above enzymes atactivities of 0.17 U/ml and 0.10 U/ml, respectively, for 18 hrs in thepresence of 0.5 mg/ml of a tribonectin purified from synovial fluid. Inall cases, the gel slices were cut through the middle of the band andwere 16 mm³ in size. All contact surfaces were carefully cleaned with50% (v/v) acetonitrile. Sequence data was entered into the BLASTGENBANK® search algorithm and matches identified.

Isolation and Culture of Human Synovial Fibroblasts

Human synovium with a normal appearance was obtained from a 30 year oldwhite male undergoing arthroscopy. Within 1 hr after surgery, thesynovial tissue explant was washed three times with Dulbecco's calcium-and magnesium-free phosphate-buffered saline (GIBCO). Pieces 2 mm³ insize were placed in Dulbecco's modification of Eagle's medium (GIBCO),supplemented with 100 U of penicillin and 100 μg of streptomycin per ml(GIBCO), containing 4 mg/ml of Clostridiopeptidase A (WorthingtonBiochemical CLS, 125-200 U/mg) sterilized through a 0.22 μm filter(Nalge). The tissue fragments were further divided with scissors in a100 mm plastic petri dish (Falcon) and incubated for 4 hrs in 20 ml ofmedium at 37° C. in a moist atmosphere of 5% carbon dioxide and 95% air.

The digest was well mixed many times by aspiration into and expulsionfrom a Pasteur pipette. An equal volume of 0.05% trypsin and 0.02% EDTAin modified Puck's Saline A (GIBCO) were added and incubation continuedfor a further hour under the same conditions. The suspension wascentrifuged 10 min at 400×g at 23° C. and washed three times each with40 ml of calcium- and magnesium-free phosphate-buffered saline. Thepellet was suspended in modified Eagle's medium (20 ml) supplementedwith 10% fetal bovine serum (Flow Laboratories), 100 U of penicillin,and 100 mg of streptomycin per ml. Two milliliters of this final mixturewere plated per 60 mm plastic petri dish (Falcon). Synovial fibroblastswere grown to confluence and cells harvested. Human skin fibroblasts(American Type Culture Collection (ATCC) Designation CCD-1099SK; ATCC,Mannassas, Va.) which served as a control were also grown and harvestedusing the above procedure.

RNA Extraction and RT-PCR Analyses

RNA from synovial and skin fibroblasts was purified by RNeasymini-columns and reagents (Qiagen, Crawley, Ltd., UK). Contaminatinggenomic DNA was removed by DNAshredder and DNase (RNase free) (Qiagen).First strand cDNA was synthesized by reverse transcription and PCRamplification using the following oligonucleotide primers.

MSF-exon 6 forward primer (SEQ ID NO: 15)5′-CCAAACCACCAGTTGTAGATGAAGC-3′ and MSF-exon 6 reverse primer (SEQ IDNO: 16) 5′-GCGGAAGTAGTCTTCTCTTTTCCATCAG-3′.These primers correspond to nucleotide position numbers 674-698 and953-926, respectively, of the human MSF gene (SEQ ID NO:2; GENBANK®accession number U70136). Thermal cycling conditions were 42° C. for 12mins., 95° C. for 10 mins., followed by 43 cycles between 94° C.×20 secsand 55-65° C.×30-90 secs. A final extension for 7 mins was at 72° C.(Perkin Elmer Biosystems).

Alternative Splice Variant of MSF is a Tribonectin

A lubricating polypeptide was purified from human synovial fluid usingstandard biochemical methods followed by affinity chromatography withpeanut agglutinin. The final fraction, which solely possessedlubricating ability, contained a product with an apparent molecularweight of 280 kDa. Components with a molecular weight in excess of 280kDa were not observed. LCMS performed on tryptic fragments from the 280kDa excised band indicated the presence of two different proteins thatmatched in the BLAST search algorithm to fibronectin precursor and MSF(GenBank Accession No. U70136). Sequences of MSF were identified fromboth native and deglycosylated lubricating polypeptides. Accordingly,the purification scheme was terminated with an anti-fibronectin columnresulting in the elimination of fibronectin as an impurity (as assayedby C18 analytical HPLC and purity plot analysis). In addition, lowermolecular weight bands at 70 and 160 kDa on SDS-PAGE were absent fromthe purified tribonectin preparation eluting from the anti-fibronectincolumn. The purified tribonectin assayed in the friction apparatus wasfound to display boundary lubricating activity similar to that of wholesynovial fluid (Table 5). By contrast, purified serum fibronectin raisedfriction indicating that synovial fluid lubricating ability was mediatedby the purified tribonectin.

TABLE 5 Friction coefficients for a tribonectin purified from humansynovial fluId and fibronectin (Mean ± SD; N = 3) LUBRICANT* μ μ (PS**⁾Δμ Tribonectin 0.047 ± .006 0.131 ± .007 −0.084 ± .004 HSF† 0.040 ± .0050.135 ± .009 −0.095 ± .011 Fibronectin 0.181 0.136 +0.045 ± .005

Tested at a concentration of 250 μg/ml in PS.

Physiological saline.

Post-mortem human synovial fluid

Furthermore, LCMS of tryptic fragments identified portions of exons 6through 9 of MSF, inclusively. Purified tribonectin reacted to peanutagglutinin indicating the presence of β(1-3)Gal-GalNAC oligosaccharidesby virtue of its purification. An increase in electrophoretic mobilitywas observed after digestion with NaNase III and O-glycosidase DS,indicating that the purified tribonectin is highly glycosylated viaO-linked oligosaccharides. The apparent molecular weight ofdeglycosylated tribonectin purified from synovial fluid was 120 kDa.

RT-PCR analysis was completed using primers specific for nucleotidesequences encoding the N-terminal end of exon 6 of MSF. RT-PCR's usinghuman synovial fibroblast RNA generated a 280 bp product, the predicteddistance between the designed primers. Similar experiments withoutreverse transcriptase did not generate this product indicating that theRNA was free of genomic DNA. Purified RNA from skin fibroblasts did notproduce any product using the same primers.

MSF was first isolated from human monocytes; a 25 kDa fragment of MSFwas found to stimulate the development of megakaryocytes. MSF precursorprotein is 1404 residues in size and constructed from 12 exons. Exon 6appears to encodes a centrally located mucin that is 940 residues inlength. Exon 6 has homology to vitronectin, exons 2 and 3 appearhomologous to somatomedin B-like regions, and exons 8, 9 are similar tohemopexin-like regions in vitronectin. Hemopexin is a serum hemescavenging protein that interacts with hyaluronate.

A tribonectin purified from synovial fluid and an articular cartilagesuperficial zone protein (SZP) purified from articular cartilage sharesequence identity with MSF but differ in their apparent molecularweights and amino acid sequences.

Other embodiments are within the following claims.

1. An isolated tribonectin comprising an amino acid sequence having atleast one but less than 76 subunits, wherein (a) each subunit comprisesat least 7 amino acids; (b) the amino acid sequence of said subunitcomprises at least one threonine and has at least 50% identity to SEQ IDNO: 3, wherein a non-identical amino acid is a conservative amino acidsubstitution; (c) said tribonectin comprises β(1-3) Gal-GalNAc moieties;and (d) at least 10% by weight of said tribonectin comprises O-linkedoligosaccharides.
 2. The tribonectin of claim 1, wherein the amino acidsequence of said subunit is SEQ ID NO:
 3. 3. The tribonectin of claim 1,wherein said tribonectin further comprises one or more repeats of theamino acid sequence of SEQ ID NO:
 4. 4. The tribonectin of claim 1,wherein said tribonectin comprises an amino acid sequence that is atleast 85% identical to amino acids 200-1140, inclusive, of the aminoacid sequence of SEQ ID NO:
 1. 5. The tribonectin of claim 4, whereinsaid tribonectin comprises amino acids 1-24 and 200-1404 of SEQ ID NO:1, wherein said tribonectin lacks amino acids 25-199 of SEQ ID NO:
 1. 6.The tribonectin of claim 4, wherein said tribonectin comprises aminoacids 1-156 and 200-1404 of SEQ ID NO: 1, wherein said tribonectin lacksamino acids 157-199 of SEQ ID NO:
 1. 7. The tribonectin of claim 4,wherein said tribonectin comprises amino acids 1-106 of SEQ ID NO: 1 and200-1404 of SEQ ID NO: 1, wherein said tribonectin lacks amino acids107-199 of SEQ ID NO:
 1. 8. The tribonectin of claim 4, wherein saidtribonectin comprises amino acids 1-25 of SEQ ID NO: 1, 67-106 of SEQ IDNO: 1, and 200-1404 of SEQ ID NO: 1, wherein said tribonectin lacksamino acids 26-66 of SEQ ID NO:
 1. 9. The tribonectin of claim 4,wherein said tribonectin is characterized as reducing the coefficient offriction between bearing articular cartilage surfaces.
 10. Thetribonectin of claim 4, wherein said tribonectin is characterized asreducing the coefficient of friction between bearing surfaces in vitro.11. The tribonectin of claim 4, wherein said tribonectin ischaracterized as reducing the coefficient of friction between bearingsurfaces in vivo.
 12. The tribonectin of claim 4, wherein saidtribonectin does not substantially increase the viscosity of a solutionto which it is added.
 13. The tribonectin of claim 1, wherein at least20% of said tribonectin is glycosylated by said O-linked oligosaccharidemoiety.
 14. The tribonectin of claim 1, wherein at least 40% of saidtribonectin is glycosylated by said O-linked oligosaccharide moiety. 15.The tribonectin of claim 1, wherein the molecular weight of saidtribonectin is in the range of 220-280 kDa.
 16. The tribonectin of claim1, wherein said tribonectin comprises a fragment of megakaryocytestimulating factor.
 17. The tribonectin of claim 4, wherein saidtribonectin comprises an amino acid sequence that is at least 95%identical to amino acids 200-1140, inclusive, of the amino acid sequenceof SEQ ID NO: 1
 18. The tribonectin of claim 17, wherein saidtribonectin comprises an amino acid sequence that is at least 98%identical to the sequence of residues 200-1140, inclusive, of SEQ IDNO:
 1. 19. The tribonectin of claim 4, wherein said tribonectincomprises the amino acid sequence of residues 200-1140, inclusive, ofSEQ ID NO:
 1. 20. The tribonectin of claim 4, wherein said tribonectincomprises an amino acid sequence that is at least 95% identical to thesequence of residues 200-1167, inclusive, of SEQ ID NO:
 1. 21. Thetribonectin of claim 4, wherein said tribonectin comprises the aminoacid sequence of residues 200-1167, inclusive, of SEQ ID NO:
 1. 22. Thetribonectin of claim 4, wherein said tribonectin comprises an amino acidsequence that is at least 95% identical to the sequence of residues200-1212, inclusive, of SEQ ID NO:
 1. 23. The tribonectin of claim 4,wherein said tribonectin comprises the amino acid sequence of residues200-1212, inclusive, of SEQ ID NO:
 1. 24. The tribonectin of claim 4,wherein said tribonectin comprises an amino acid sequence that is atleast 95% identical to the sequence of residues 200-1263, inclusive, ofSEQ ID NO:
 1. 25. The tribonectin of claim 4, wherein said tribonectincomprises the amino acid sequence of residues 200-1263, inclusive, ofSEQ ID NO:
 1. 26. The tribonectin of claim 4, wherein said tribonectinlacks the amino acid sequence of residues 1-24, inclusive, of SEQ IDNO:
 1. 27. The tribonectin of claim 4, wherein said tribonectin lacksthe amino acid sequence of residues 67-104, inclusive of SEQ ID NO: 1.28. The tribonectin of claim 1, wherein said tribonectin lubricates amammalian joint.
 29. The tribonectin of claim 28, wherein said joint isan articulating joint of a human, dog, or horse.
 30. The tribonectin ofclaim 1, wherein said tribonectin inhibits tissue adhesion formation ina mammal.
 31. The tribonectin of claim 1, wherein the β(1-3) Gal-GalNAcmoiety further comprises a sialic acid (NeuAc) cap.
 32. A compositioncomprising the tribonectin of claim 1 and a pharmaceutically acceptablecarrier.
 33. The composition of claim 32, wherein said composition is inthe form of a membrane, foam, gel, or fiber.
 34. The composition ofclaim 32, wherein said composition is formulated for timed release ofsaid tribonectin.
 35. The composition of claim 32 further comprisinghyaluronic acid.